A. Field of Invention
The present invention relates to derivatives of succinic and glutaric acids and analogs thereof, useful as inhibitors of PHEX. More specifically, the present invention relates to derivatives of succinic and glutaric acids and analogs thereof useful for promoting faster regeneration of bone mass after bone fractures, implantation of orthopedic and dental prostheses, or after loss of bone mass as a consequence of bone diseases such as osteoporosis.
B. Related Art
The PHEX gene (formerly PEX; Phosphate regulating gene with homologies to endopeptidases on the X chromosome) was identified by a positional cloning approach as the candidate gene for X-linked hypophosphatemia (XLH) (The Hyp Consortium, 1995). XLH is a Mendelian disorder of phosphate homeostasis characterized by growth retardation, rachitic and osteomalacic bone disease, hypophosphatemia, and renal defects in phosphate re-absorption and vitamin D metabolism (Tenenhouse and Econs, 2001).
Several groups have cloned and sequenced the human and mouse PHEX/Phex cDNAs (PHEX/Phex refers to the human and mouse genes, respectively) (Du et al., 1996; Lipman et al., 1998; Grieff et al., 1997; Beck et al., 1997; Guo and Quarles, 1997; Strom et al., 1997). Amino acid sequence comparisons have demonstrated homologies between PHEX/Phex protein and members of the neutral endopeptidase family, as previously observed in the partial sequence of the candidate PHEX gene (The HYP Consortium, 1995). The peptidases of the neutral endopeptidase family are zinc-containing type II integral membrane glycoproteins having a relatively short cytoplasmic N-terminal region, a single transmembrane domain, and a long extracytoplasmic domain containing the active site of the enzyme (Turner and Tanzawa, 1997).
Much of the present knowledge about XLH has been obtained from studies of the Hyp mouse, which harbours a large deletion of the Phex gene (Beck et al., 1997) and which has been used as an animal model of the human disease (Tenenhouse, 1999). In particular, these animals show increased renal phosphate excretion due to a down-regulation of the Npt2 phosphate transporter, which is necessary for the re-absorption of phosphate from the nephron. The serum concentration of 1,25(OH)2D3 (calcitriol) was found to be the same in Hyp mice as in normal littermates. However, the Hyp kidney showed an accelerated degradation of the vitamin D metabolite to 1,24,25(OH)3D3, a metabolite with reduced activities. In the presence of a phosphate rich diet, Hyp mice were shown to experience an increase in serum 1,25(OH)2D3 and a drop in C-24 oxidation products, in contrast to normal mice which experienced no such changes. The renal disorder in vitamin D metabolism in Hyp mice appears to be secondary to the phosphate disorder.
The mechanism by which loss of PHEX function elicits the observed bone and renal abnormalities in XLH patients is not clear. There are no data suggesting the presence of PHEX/Phex mRNA in the kidney (Du et al., 1996; Beck et al., 1997; Grieff et al., 1997). However, considering the similarities between the PHEX protein and the other members of this metallopeptidase family, it has been speculated that PHEX may regulate renal phosphate reabsorption by controlling the activity of a circulating factor. It was demonstrated that the inhibition of Na-dependent phosphate transport in cultured renal cells can be achieved by a factor in conditioned medium from cultured osteoblasts derived from Hyp mice (Lajeunesse et al. 1996; Nesbitt et al., 1999).
Phosphaturic activity(ies) have also been discovered in tumors from patients with tumor-induced osteomalacia (TIO, also known as oncogenic hypophosphatemic osteomalacia), an acquired renal phosphate wasting disorder with the phenotypic features of XLH (Tenenhouse and Econs, 2001). The term “phosphatonin” was designated to depict the phosphaturic tumor factor(s) (Econs and Drezner, 1994) and although the exact nature of “phosphatonin” remains to be determined, several candidates have been proposed (Schiavi and Moe, 2002). It has been shown that mutations in the FGF-23 gene which encodes a novel growth factor, fibroblast growth factor-23 (FGF-23), is responsible for Autosomal Dominant Hypophosphatemic Rickets (ADHR), an inherited disorder that resembles XLH and TIO (ADHR Consortium, 2001). Moreover, it has been demonstrated that overexpression of FGF-23 in animal models elicits renal phosphate wasting, a reduction in serum phosphate levels and osteomalacia (Shimada et al., 2001). Of interest are the findings that FGF-23 is overexpressed in tumors from patients with TIO (Jan de Beur et al., 2002) as well as in XLH patients (Jonsson et al., 2002). In addition to FGF-23, other proteins such as Frizzled-related protein 4 (FRP-4) (Jan de Beur et al., 2002) and MEPE (matrix extracellular phosphoglycoprotein) (Rowe et al., 2000) are overexpressed in TIO tumors.
PHEX/Phex mRNA has been detected in bones by Northern blot hybridization and in other adult and fetal tissues such as lungs, liver, muscles, and ovaries by RT-PCR and RNase protection assays (Du et al. 1996; Beck et al., 1997). In situ hybridization performed on sections of embryos and newborn mice showed the presence of Phex mRNA in osteoblasts and odontoblasts (Ruchon et al., 1998). Phex gene expression was detectable on day 15 of embryonic development, which coincides with the beginning of intracellular matrix deposition in bones. Moreover, Northern blotting analysis of total RNA from calvariae and from teeth of 3-day-old and adult mice showed that the abundance of the Phex transcript had decreased in adult bones and in non growing teeth. This result was confirmed when the presence of the Phex protein in newborn adult bones was investigated by Western blotting using a monoclonal antibody raised against the human PHEX. Immunohistochemical studies on a 2 month-old mouse showed exclusive labeling of mature osteoblasts and osteocytes in bones, and of odontoblasts in teeth (Ruchon et al., 2000). These results suggest that PHEX/Phex is important in both the development and maintenance of mineralization in these tissues. This hypothesis was supported by evidence of intrinsic abnormalities in osteoblasts from Hyp mice (Ecarot et al., 1992; Karaplis 2003). PHEX might thus be involved in the control of bone metabolism, both indirectly at the kidney level by controlling renal phosphate reabsorption, and directly at the bone level by inactivating a trophic peptide factor controlling either osteoblast or osteoclast functions or both.
Osteogenesis is a complex biological process that includes proliferation and differentiation of bone-forming cells (osteoblasts), synthesis of an organic matrix composed mainly of type I collagen, and mineralization of the organic matrix by deposition of hydroxyapatite crystals. Various technologies have been developed to stimulate osteogenesis for bone regeneration in osseous reconstructive surgery. These include the use of bone morphogenetic proteins (BMPs) as osteogenic agents, mostly in combination with a solid support such as metal meshes (Vehof et al., 2001), atelopeptide type I collagen (Ikeuchi et al., 2002) or hydroxyapatite (Yoshida et al., 1999). Hydroxyapatite is an osteoconductive material that maintains an original biocompatible form. During reconstruction of bone defects, its osteoconduction can be enhanced with osteogenic agents such as BMPs. More recently, evidence has emerged that novel and still poorly characterized peptides might also be useful for stimulating osteogenesis. These peptides are thought to be involved in poorly characterized pathways regulating bone mineralization. It is surmised that these pathways could be under the control of PHEX.
There thus remains a need to develop selective PHEX inhibitors to control phosphate metabolism and which can be used as osteogenic agents, as well as methods of administering the PHEX inhibitors.
The present invention seeks to meet these and other needs. The present description refers to a number of documents, the contents of which is herein incorporated by reference in their entirety.